EP3154567B1 - Expansion de lymphocytes avec une composition de cytokine pour immunothérapie cellulaire active - Google Patents

Expansion de lymphocytes avec une composition de cytokine pour immunothérapie cellulaire active Download PDF

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EP3154567B1
EP3154567B1 EP15737995.9A EP15737995A EP3154567B1 EP 3154567 B1 EP3154567 B1 EP 3154567B1 EP 15737995 A EP15737995 A EP 15737995A EP 3154567 B1 EP3154567 B1 EP 3154567B1
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cells
lymphocytes
tumor
expansion
cell
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EP3154567A1 (fr
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Markus Maeurer
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Polybiocept GmbH
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Definitions

  • the present invention relates to active cellular immunotherapy including a method of preparing a population of clinically relevant lymphocytes using a composition of predefined cytokines.
  • the invention further relates to the composition of cytokines and the generated clinically relevant lymphocytes.
  • Cancer remains to be one of the most common causes of death in the developed countries. For example in the United States and Germany it is the second most common cause of death with a mortality of 560,000 (2009) and 218,000 (2010), respectively. Survival rates remain poor for many cancers despite improvement in the ability to detect and treat this constellation of diseases.
  • pancreatic cancer is the fourth leading cause of cancer death in the United States and Sweden without signs of improvement.
  • pancreatic cancer is the third leading cause of cancer death in the United States and Sweden without signs of improvement.
  • the medium survival is about six months. Only about 15 to 20 % of the patients have a resectable tumor and therefore a potentially curable disease.
  • pancreatic cancer is a systemic disease that requires early and systemic intervention.
  • pancreatic cancer is highly chemotherapy resistant and radiation resistant.
  • the biology of pancreatic cancer is associated with both local and systemic immunosuppression enabling tumor progression and metastasizing.
  • glioblastoma which is the most frequent and progressive glioma with an incidence of 2-3/100.000 in the United States.
  • Glioblastoma make up 12 to 15 % of all intracranial and 50 to 60 % of histiocytic tumors.
  • New treatment regimens have increased a medium overall survival (14.6 months with radio plus temozolomide as compared to 12.1 months with radio therapy alone). So far intents to develop robust and clinically effective immunotherapeutic protocols have been frustrating for patients with glioblastoma or patients with pancreatic cancer.
  • One approach for treating such cancers is to overcome the tumor induced suppression and/or to induce anti-tumor directed cellular and humoral immune responses.
  • Cancer immunotherapies can be either passive or active. Passive therapy is based on the adoptive transfer of immunomodulators including cytokines, tumor specific antibodies or immune cells. These substances or cells are then administered to the patient to initiate an anti-tumor action. In general these therapies do not generate immunologic memory and therefore require chronic infusion based treatment. Active immunotherapies, on the other hand, stimulate the patient's immune system with the intent of promoting an antigen specific anti-tumor effect using the body's own immune cells. In addition active immunotherapies seek to create durable anti-tumor response that can protect against minimal residual disease and tumor recurrences.
  • T-cells directed against tumors have been achieved in patients with melanoma (2, 3).
  • a landmark article showed recently that the best and durable response in cancer treatment is achieved if the patient's own T-cells are directed against the patient's own tumor cells, i.e. the patient's own "private" mutations (4).
  • Such promising results were also obtained for patients with epithelial tumors, i.e. by adaptive transfer of T-cells targeting mutant epitopes in epithelial cancer (5).
  • TIL tumor infiltrating lymphocytes
  • TIL Minimally cultured TIL appears to provide the most effective phenotype and profile for clinical use (11).
  • the most successful approach up to date has been the use of autologous ex vivo activated T-cells that were grown in 24 well plates, tested for immune effector functions and further expansion using IL-2, allogeneic feeder cells and OKT3 (9, 12, 13).
  • CD4+ or CD8+ tumor antigen (TAA) directed T-cells have been generated under GMP conditions from peripheral blood and formulated to subsequent treatment of patients. This has been achieved with autologous CD4+ T-cells (14- 16) or CD8+ T-cells(17), some directed against the NY-ESO-1 antigen(18), which is also possible in PBMCs from healthy patients without cancer, since sufficient T-cell precursors are present in the peripheral circulation.
  • Various methods for expansion of T-cells generating CD8+ T-cell clones for target directed therapy (19) have been described. This if great interest since clonal repopulation of the patient's immune system with anti-tumor lymphocytes has been shown to induce cancer regression but also autoimmunity (20).
  • the growth medium formulation may also be relevant for successful active immunotherapy.
  • starvation impacts on T-cell mediated immune responses and may induce starvation- induced immunosuppression, yet also the expansion of certain T-cell subsets This mechanism appears to be mediated by Leptin (21) which modulates also B-cell development and subsequent B-cell responses (22).
  • Leptin i.e. the GCN2 in dendritic cells
  • cytokine-driven T-cell expansion are dependent on exogenous amino acids and that cytokines, i.e. IL-7, upregulated genes associated with amino acid transporter expression. Tailoring growth media requirements will therefore be shaped by the respective cytokine cocktail used for T- cell expansion (24) as well as for amino acids in the medium; both factors will impact on T-cell maturation and differentiation which is clinically meaningful.
  • WO 2008/066609 A1 describes a method for carrying out adoptive immunotherapy in a primate subject by administering to the subject a cytotoxic T-lymphocyte (CTL) preparation in a treatment effective amount.
  • CTL cytotoxic T-lymphocyte
  • T-cells are expanded with a starting concentration of 10 6 cells/well in T-cell media alone or media with IL-2, IL-15 or IL-7.
  • WO 2007/071390 A1 and WO 2007/071409 A1 relate to an immunotherapeutic method for treating patients suffering from malignant melanoma by administering expanded tumour-reactive CD4+ helper and/or CD8+ T-lymphocytes obtainable from one or more sentinel or metinel lymph nodes draining a malignant melanoma or a metastasis arising from malignant melanoma.
  • the described expansion method comprises a first phase using IL-2 and a second phase using IL-4, IL-5, IL-10 and/or TGF-beta.
  • Huarte et al. 2009 addresses the generation of antigen specific central memory T-cells.
  • Huarte et al. describe a protocol for the production of a lymphocyte population enriched for tumour specific T-cells.
  • healthy donor or melanoma patient lymphocytes are stimulated with autologous matured dendritic cells (mDC) pulsed with a pool of class-I and class-II melanoma antigens in the presence of IL-2 or IL-15 plus IL-21.
  • mDC autologous matured dendritic cells
  • Santegoets et al., 2013 is a further scientific article relating to adoptive cell transfer of tumour infiltrating lymphocytes.
  • the experiments start from a rapid expansion method (REM) based on the established "Rosenberg method" with IL-2, irradiated allogeneic feeder cells and CD-3 ligation via an anti-CD-3 antibody.
  • REM rapid expansion method
  • the authors of Santegoets et al. have compared the effects of the individual cytokines IL-2, IL-15 and IL-21 for the expansion and activation of TIL from single cell suspensions of small cell lung cancer.
  • WO 2010/056144 A2 relates to isolated populations of cells comprising FoxP3+ natural killer T-cells (NK T-cells), methods of generating FoxP3+ NK T-cells and methods for suppressing the immune this response in specific organs, including the liver and the lungs.
  • the NK T-cells are cultured with TGF ⁇ and one or more NK T-cell stimulants.
  • the culture medium may contain IL-2 and it is stated that in some embodiments, the population of cells may also be contacted with IL-7, IL-15 and/or IL 21.
  • T-cells are CD8+ and central memory cells, defined by CD45RA-CCR7+, defined ex vivo from patients responding to T-cell based therapy.
  • the phenotype of such T-cells is determined by the ex vivo expanded T-cell population, as well as by host factors after adoptive transfer.
  • a diverse population of T-cells targeting cancer cells may be advantageous for effective immune responses, including long-term memory T-cells, yet also T-cells that can immediately react to (cancer) target cells and produce anti-tumor directed immune responses, including terminally differentiated T-cells that express cytolytic molecules, such as granzyme and perforin (25, 26).
  • Long term-memory immune memory is in part determined by the increased proliferation potential and half-life that can be measured by the telomere length (27, 28).
  • activation/exhaustion markers i.e. LAG-3, PD-1 and or 4-1BB on T-cells that may indicate a higher change for T-cell exhaustion and loss of function, and also an enrichment of tumor-antigen specific T-cells (which tend to be PD1+, and/or LAG-3, 4-1BB+ (29)).
  • NY-ESO-1 was known to be an eligible target for tumor- antigen specific T-cells.
  • NY-ESO-1 is a cancer testing antigen (36, 37) and expressed in a high number of tumors. For example, at Karolinska 50 glioblastoma lesions were screened for NY-ESO-1 protein expression and it was found that 35% of the grade 3 and 4 GB are positive for NY-ESO-1. Screening of pancreatic cancer lesions showed a lower number of NY-ESO-1 protein + cancer lesions in the range of 20%, particularly in metastatic lesions.
  • NY-ESO-1 for the expansion of tumor-reactive T-cells from peripheral blood appears to be a 'safe choice' of target, since NY-ESO-1 appears to be expressed only in malignant cells and testis without overt 'off-target' reactivity in anti-NY-ESO-1 directed T-cells (36). This is of great interest since clonal repopulation of the patients immune system with antitumor lymphocytes has been shown to induce cancer regression, and autoimmunity(20), a potential risk. NYESO-1 has been tested in a number of studies as a potential target in GB, as well as in GB-stem cells (38), along with the use of DNA- methylating agents to increase NY-ESO-1 reactivity (39, 40).
  • the present invention is inter alia based on the finding that a composition comprising cytokines interleukin-2 (IL-2), interleukin-15 (IL-15) and/or interleukin-21 (IL-21) leads to a superior stimulation and expansion of lymphocytes in particular clinically relevant lymphocytes.
  • IL-2 interleukin-2
  • IL-15 interleukin-15
  • IL-21 interleukin-21
  • the invention provides a composition for in vitro expanding lymphocytes comprising interleukin 2 (IL-2), interleukin 15 (IL-15) and interleukin 21 (IL-21), wherein the composition is in liquid form, the concentration of IL-2 in the liquid composition is in the range of 500 to 2000 U/ml, the concentration of IL-15 is in the range of 0.1 to 100 ng/ml and wherein the concentration of IL-21 is in the range of 0.1 to 100 ng/ml.
  • IL-2 interleukin 2
  • IL-15 interleukin 15
  • IL-21 interleukin 21
  • the present invention provides a method of preparing a population of clinically relevant lymphocytes, comprising the steps of:
  • the method of the second aspect of the invention leads to the formation of population of lymphocytes, which includes a population of clinically relevant lymphocytes.
  • the population of clinically relevant lymphocytes obtained with the method according to the second aspect of the invention is in particular advantageous for cellular immunotherapy.
  • the invention provides a composition comprising a population of clinically relevant lymphocytes according to the third aspect of the invention for use in the medical treatment, in particular for treating and preventing an infectious disease, an autoimmune disease or a tumor disease.
  • the inventors have found that a combination of the interleukins IL-2, IL-15 and IL-21 provides significant improvements for immunotherapy with lymphocytes.
  • One major advantage is that the expansion and stimulation of lymphocytes derived from a patient with the composition of a combination of IL-2, IL-15 and IL-21 specifically favors the generation of lymphocytes, in particular T-cells, which are clinically relevant.
  • clinically relevant lymphocytes are specific for and interact with clinically relevant antigens.
  • Chronic relevant lymphocytes are also referred to as antigen-edited lymphocytes.
  • the term clinically relevant is also used for subgroups of lymphocytes.
  • Particularly preferred clinically relevant lymphocytes are clinically relevant T-cells or antigen-edited T-cells.
  • Clinically relevant antigens are antigens involved in a disease. Accordingly, clinically relevant antigens can be tumor-associated antigens TAA, pathogen associated antigens (PAA) or autoantigens. Tumor-reactive lymphocytes are specific for and interact with TAAs. Infectious disease reactive lymphocytes are specific for and interact with PAAs and autoimmune disease reactive lymphocytes are specific for and interact with autoantigens.
  • TAA tumor-associated antigens
  • PAA pathogen associated antigens
  • autoantigens Tumor-reactive lymphocytes are specific for and interact with TAAs.
  • Infectious disease reactive lymphocytes are specific for and interact with PAAs
  • autoimmune disease reactive lymphocytes are specific for and interact with autoantigens.
  • an "antigen" is any structural substance which serves as a target for the receptors of an adaptive immune response, TCR or antibody, respectively.
  • Antigens are in particular proteins, polysaccharides, lipids and substructures thereof such as peptides. Lipids and nucleic acids are in particular antigenic when combined with proteins or polysaccharides.
  • Pathogen associated antigens refer to parts, such as capsules, cell walls, flagella and toxins of pathogens such as bacteria, viruses and other microorganisms.
  • Autoantigens are usually peptides, oligopeptides, polypeptides or complexes of proteins from an individual that are recognized by the immune system of the same individual. This effect usually leads to an autoimmune disease.
  • TAA Tuor associated antigens
  • TAA tumor associated antigens
  • TAA includes “tumor-specific antigens” which are found only on the surface of tumor cells, but not on the surface of normal cells.
  • the method according to the invention provides an easy protocol for the expansion of clinically relevant lymphocytes. It is in particular advantageous in comparison to the protocols in the state of the art as no dendritic cells are required. Moreover, the inventors could show that the resulting lymphocyte population after expansion with the cytokine cocktail of IL-2, IL-15 and IL-21 contains a composition of lymphocytes that is advantageous for clinical application. For example, the composition has a high percentage of T H1 helper T-cells and almost no T H2 helper T-cells. A further advantage is that there is no significant expansion of regulatory T-cells which might cause a suppression of the therapeutic action of the expanded lymphocyte population.
  • the invention provides a composition for in vitro expanding lymphocytes comprising interleukin 2 (IL-2), interleukin 15 (IL-15) and interleukin 21 (IL-21), wherein the composition is in liquid form, the concentration of IL-2 in the liquid composition is in the range of 500 to 2000 U/ml, the concentration of IL-15 is in the range of 0.1 to 100 ng/ml and wherein the concentration of IL-21 is in the range of 0.1 to 100 ng/ml.
  • IL-2 interleukin 2
  • IL-15 interleukin 15
  • IL-21 interleukin 21
  • IL-2, IL-15 and IL-21 are members of the cytokine family each of which has a four alpha helix bundle.
  • IL-2 has key roles in key functions of the immune system, tolerance and immunity, primarily via its direct effects on T-cells. IL-2 induces T-cells proliferation and differentiation into effector and memory T-cells.
  • IL-15 is a cytokine that is structurally similar to IL-2. Like IL-2, IL-15 binds to and signals through a complex composed of the IL-2/IL-15 receptor beta chain. IL-15 induces a T-cell activation and proliferation in particular of CD8+ T-cells (30) and also provides survival signals to maintain memory cells in the absence of antigens, favored CD8+ T-cells and activates monocytes. IL-15 appears to drive rather proliferation of immune effector T-cells, along with the protection from inhibition of tumor-associated immunosuppression (31).
  • IL-21 is a cytokine that has potent regulatory effects on cells of the immune system, including natural killer (NK) cells and cytotoxic T-cells.
  • IL-21 enriches central memory type T-cells with a CD28+ CD127hi CD45RO+ phenotype and enhances the cytotoxity of cytotoxic T-cells.
  • IL-21 may keep T-cells in their early phase of differentiation and maturation (35).
  • composition of a combination of IL-2, IL-15 and IL-21 is also referred to as "the cytokine cocktail”.
  • interleukin 2 refers to human IL-2 as defined by SEQ ID NO: 1 and functional equivalents thereof.
  • Functional equivalents of IL-2 include relevant substructures or fusion proteins of IL-2 that remain the functions of IL-2.
  • the definition IL-2 comprises any protein with a sequence identity to SEQ ID NO: 1 of at least 80 %, preferably at least 90 %, more preferably at least 95 %, most preferably at least 98 %.
  • Recombinant human IL-2 produced in E. coli as a single, non-glycosylated polypeptide chain with 134 amino acids and having a molecular mass of 15 kDa is commercially available in lyophilized form from Prospec as CYT-209.
  • interleukin 15 or "IL-15” refer to human IL-15 and functional equivalents thereof.
  • Functional equivalents of IL-15 include relevant substructures or fusion proteins of IL-15 that remain the functions of IL-15.
  • the definition IL-15 comprises any protein with a sequence identity to SEQ ID NO: 2 of at least 80 %, preferably at least 90 %, more preferably at least 95 %, most preferably at least 98 %.
  • coli as a single, non-glycosylated polypeptide chain with 114 amino acids (and an N-terminal Methionine) and having a molecular mass of 12.8 kDa is commercially available in lyophilized form from Prospec as CYT-230.
  • interleukin 21 refers to human IL-21 and functional equivalents thereof. Functional equivalents of IL-21 included relevant substructures or fusion proteins of IL-21 that remain the functions of IL-21. Accordingly the definition IL-21 comprises any protein with a sequence identity to SEQ ID NO: 3 of at least 80 %, preferably at least 90 %, more preferably at least 95 %, most preferably at least 98 %.
  • Recombinant human IL-21 produced in E. coli as a single, non-glycosylated polypeptide chain with 132 amino acids and having a molecular mass of 15 kDa is commercially available in lyophilized form from Prospec as CYT-408.
  • a "peptide” as used herein may be composed of any number of amino acids of any type, preferably naturally occurring amino acids, which, preferably, are linked by peptide bonds.
  • a peptide comprises at least 3 amino acids, preferably at least 5, at least 7, at least 9, at least 12, or at least 15 amino acids.
  • there is no upper limit for the length of a peptide preferably, a peptide according to the invention does not exceed a length of 500 amino acids, more preferably it does not exceed a length of 300 amino acids; even more preferably it is not longer than 250 amino acids.
  • peptide includes “oligopeptides”, which usually refer to peptides with a length of 2 to 10 amino acids, and “polypeptides” which usually refer to peptides with a length of more than 10 amino acids.
  • protein refers to a peptide with at least 60, at least 80, preferably at least 100 amino acids.
  • fusion protein relates to proteins created through the joining of two or more genes, cDNAs or sequences that originally coded for separate proteins/peptides.
  • the genes may be naturally occurring in the same organism or different organisms or may synthetic polynucleotides.
  • sequence identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".
  • sequence identity the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm ( Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 ) as implemented in the Needle program of the EMBOSS package ( EMBOSS: The European Molecular Biology Open Software Suite, Rice et a/., 2000, Trends Genet. 16: 276-277 ), preferably version 3.0.0 or later.
  • the optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • transitional term "comprising”, which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.
  • the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim, except for impurities ordinarily associated therewith.
  • the transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention.
  • a 'consisting essentially of' claim occupies a middle ground between closed claims that are written in a 'consisting of' format and fully open claims that are drafted in a 'comprising' format.”
  • “Expansion” or “clonal expansion” as used herein means production of daughter cells all arising originally from a single cell. In a clonal expansion of lymphocytes, all progeny share the same antigen specificity.
  • the composition according to the first aspect comprises three types of cytokines. Further cytokines may interfere with the expansion results provided by the composition according to the invention.
  • the composition of the first aspect of the invention may contain more cytokines in addition to IL-2, IL-15 and IL-21.
  • cytokines examples are IL-1beta, IL-4, GM-CSF, IL-12, IL-8, IL-17, TNF ⁇ , IL-32.
  • IL-1 ⁇ is involved in priming, the differentiation into effector B-cells or T-cells upon first contact with the specific antigen.
  • IL-4 and GM-CSF are involved in dendritic cell stimulation and/or priming.
  • IL-12 is involved in T H1 responses.
  • IL-18 stimulates ⁇ -T-cells.
  • IL-17 and TNF ⁇ act pro-inflammatory.
  • IL-32 also acts pro-inflammatory favoring long-term protective immune responses.
  • the composition of first aspect is in liquid form.
  • the composition is a cell culture medium.
  • any known cell culture medium is possible.
  • Non-limiting examples of cell culture media are a synthetic medium, a medium derived from serum, plasma or whole blood or any combination thereof.
  • the concentration of IL-2 in the liquid composition is in the range of500 to 2000 U/ml .
  • the International Unit (U) is the standard measure for an amount or IL-2. It is determined by its ability to induce the proliferation of CTLL-2 cells. More preferably, the concentration of IL-2 is in the range of 800 to 1100 U/ml. As shown in the examples optimal results were achieved with a concentration of about 1000 U/ml.
  • the concentration of IL-15 is in the range of 0.1 to 100 ng/ml.
  • the concentration range follows the same rational as for IL-2.
  • a concentration below 0.1 ng/ml is believed not to have any significant effect on the cells.
  • a concentration above 100 ng/ml might have a cytotoxic effect.
  • the concentration of IL-15 is in the range of 2 to 50 ng/ml, more preferably in the range of 5 to 20 ng/ml.
  • the most preferred concentration is about 10 ng/ml.
  • the concentration of IL-21 is in the range of 0.1 ng/ml to 100 ng/ml, preferably in the range of 2 to 50 ng/ml, more preferably in the range of 5 to 20 ng/ml.
  • any of these concentration ranges of one of the cytokines can be combined with any of concentration ranges of the other cytokines.
  • the combination comprises IL-2 in a concentration of 800 to 1000 U/ml and IL-15 and IL-21 in a concentration of 5 to 20 ng/ml.
  • the composition comprises IL-2 in a concentration of about 1000 U/ml and IL-15 and IL-21 in a concentration of about 10 ng/ml.
  • composition of IL-2, IL-15 and IL-21 is in particular beneficial for promoting the expansion of clinically relevant lymphocytes in a composition of lymphocytes, in particular a patient's sample.
  • the inventors have developed a method for preparing specifically clinically relevant lymphocytes from a patient's sample.
  • composition of IL-2, IL-15 and IL-21 is in particular beneficial for promoting the expansion of clinically relevant lymphocytes in a composition of lymphocytes, in particular a patient's sample.
  • the inventors have developed a method decreasing the frequency of PD1 and LAG3 + T-cells, where PD1 and/or LAG3 expression serves as a marker for T-cell exhaustion and not as marker for antigen-experienced T-cells.
  • composition of IL-2, IL-15 and IL-21 is in particular beneficial for promoting the expansion of clinically relevant lymphocytes in a composition of lymphocytes, in particular a patient's sample.
  • the inventors have developed a method increasing the frequency of 4-1 BB expression as a marker for antigen-experienced T-cells.
  • the invention provides a method of preparing a population of clinically relevant lymphocytes comprising the steps of
  • cytokines IL-2, IL-15 and IL-21 are used in the concentrations defined above.
  • the method can be used for generating a population of tumor reactive lymphocytes, autoimmune disease reactive lymphocytes or infectious disease reactive lymphocytes.
  • the lymphocyte population generated by the method of the second aspect is a population of tumor reactive lymphocytes.
  • the lymphocytes in general will comprise a variety of different lymphocytes.
  • these lymphocytes may be lymphocytes present that have the right receptor to interact with a clinically relevant antigen, in particular a tumor associated antigen, an infectious disease associated antigen or an autoimmune disease associated antigen.
  • a clinically relevant antigen in particular a tumor associated antigen, an infectious disease associated antigen or an autoimmune disease associated antigen.
  • this clinically relevant lymphocyte in particular is strongly expanded.
  • other lymphocytes that do not have the specificity for the clinically relevant antigen also are expanded in the method according to the invention.
  • the outcome of culturing the cells from the body sample with the composition comprising IL-2, IL-15 and IL-21 leads to the formation of a population of lymphocytes which includes the population of clinically relevant lymphocytes.
  • the determination of the presence of clinically relevant lymphocytes in the cultured sample is a possible but not necessary step of the method according to the second aspect of the invention.
  • the steps useful to verify the expanded lymphocytes population indeed can be used as a therapeutic.
  • the body sample can be taken from any part of the body that contains lymphocytes.
  • body samples are peripheral blood, cord blood, bone marrow, lymph nodes, liver pleural effusion, thorax, abdominal cavity, synvial fluid, peritoneum, retroperitoneal space, thymus, and tumor.
  • TIL tumor infiltrating lymphocytes
  • TIL is any kind of lymphocyte that is located in, on or around a tumor.
  • TIL is any kind of lymphocyte that is located in and around a tumor.
  • the TIL may have experienced tumor-associated antigens. Accordingly, clinically relevant lymphocytes, in particular tumor reactive lymphocytes can be expanded with the method according to the invention without expansion antigen.
  • a sample from peripheral blood is also referred to peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • different body samples may be preferred.
  • the term "mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.
  • the mammal from which the body sample is obtained is a human.
  • the mammal may have a tumor disease, or be at risk of developing a tumor disease.
  • the risk of developing a tumor disease includes a high risk, a moderated risk and a low risk.
  • a mammal in such a pre-malignant condition for example has a premalignant lesion which is morphologically atypical tissue which appears abnormal under microscopic examination and in which cancer is more likely to occur as in its apparently normal counterpart.
  • the mammal may have an infectious disease or be at risk of developing an infectious disease.
  • the risk of developing an infectious disease includes a high risk, a moderated risk and a low risk.
  • the mammal may also have an autoimmune disease or be at risk of developing and autoimmune disease.
  • the risk of developing an autoimmune disease includes a high risk, a moderated risk and a low risk.
  • a high risk to develop intracellular infections CMV, EBV, TB, HPV
  • IFN ⁇ receptor defects e.g. IL-12 or IFN ⁇
  • An intermediate risk would be immune-suppression using corticosteroids or treatment of patients with anti-TNF ⁇ or TNF ⁇ -receptor directed reagents.
  • a low risk could be the co-infection with other pathogens or the temporary decrease of immune-competence in the course of major surgery.
  • Similar examples are different clinical presentations - in association with genetic markers - of Multiple Sclerosis, rare neurological diseases, e.g. narcolepsy, Rheumatoid Arthritis, as well as chronic autoimmune-diseases associated with the gastrointestinal system.
  • peripheral blood or the tumor itself are peripheral blood or the tumor itself.
  • lymphocytes from peripheral and tumor can be treated with a method according to the invention to develop strong antitumor properties.
  • the disease is an autoimmune disease the preferred body sample is peripheral blood.
  • the disease is an infectious disease the preferred body sample is also peripheral blood.
  • clinically relevant lymphocytes can be expanded from peripheral blood in these cases. Without being bound to theory it is assumed that the peripheral blood contains lymphocytes that have been in contact with the clinically relevant antigen, e.g. on the tumor or the infection.
  • Culturing of the body sample in vitro to expand and/or stimulate lymphocytes comprise sub-steps.
  • the in vitro culturing comprises a first expansion step comprising an incubation in culture medium comprising IL-2, IL-15 and IL-21 until lymphocytes become detectable.
  • Detectable means that the lymphocytes, for example, become visible, in particular by microscopy. Lymphocytes usually become detectable using standard light microscopy upon reaching a concentration of 5 x 10 3 cells/ml.
  • the detection of the lymphocytes may include any method known in the art that is eligible to detect the presence of lymphocytes above a certain threshold.
  • the first expansion step has the purpose of gently inducing cell proliferation together with a stimulation of the cells by the cytokine cocktail.
  • the time of incubation of the first expansion step is in the range of 6 hours to 180 days.
  • the large range of incubation time is first of all due to the fact that samples from different donors may behave very differently.
  • the lymphocytes from different body samples have very different growth rates. For example lymphocytes derived directly from the tumor of a glioblastoma or a pancreas cancer grow very differently. From pancreas cancer derived lymphocytes are already detectable within two to five days. Lymphocytes derived from glioblastoma are only detectable after one to two weeks. Accordingly, lymphocytes from other body samples may take even longer to become detectable.
  • the incubation time of the first expansion step is in the range of 4 days to 10 days. It was shown that with peripheral blood cells, incubation times about 7 days are particularly beneficial for the outcome of other expansion. However, as mentioned above, depending on the sample an expansion of only 4 days may be enough or on the other hand about 10 days or more may be necessary. Due to the good results with PBMCs shown in the examples an incubation time in the range of 6 to 8 days, in particular about seven days, is preferred.
  • the culture medium of the first expansion step comprises at least one expansion antigen.
  • the expansion antigen is a known clinically relevant antigen or a fragment, a mutant or a variant thereof.
  • a "mutant" is defined as an amino acid sequence that differs from the reference sequence by an insertion, deletion or replacement of at least one amino acid.
  • the expansion antigen is preferably selected from TAA, PAA and autoantigen.
  • the method according to the invention comprises multiple copies of an expansion antigen.
  • An increased number of copies of an expansion antigen leads to increased expansion rate of a clinically relevant lymphocyte, in particular T-cells.
  • the culture medium of the first expansion step comprises multiple expansion antigens.
  • the multiple expansion antigens include a known clinically relevant antigen and a one or more mutants of the clinically relevant antigen.
  • the antigen itself also the MHC class I/peptide presenting the antigen, in particular peptide can be mutated.
  • the use of one or more wild type, variant or mutants of the clinically relevant antigen or MHC class I molecules as expansion antigens leads to a diverse set of lymphocytes, in particular T-cells reactive against the nominal clinically relevant antigen.
  • the body sample is tumor and no expansion antigen is used in culturing.
  • the tumor tissue is preferably washed twice before separating the cells in the tumor sample.
  • the culture medium of the first expansion step comprises multiple expansion antigens.
  • the T-Cell product responds in a more diverse T-Cell receptor repertoire as demonstrated by V ⁇ -usage associated with the use of the stimulating antigen(s).
  • the culture medium of the first expansion step comprises multiple expansion antigens.
  • the stimulation of T-Cells from peripheral blood results in T-Cell recognizing diverse epitopes, defined by cytoxicity.
  • T-Cells are selected from the blood and stimulated with such antigens, they result in diverse epitopes of the antigen being recognized.
  • the choice of expansion antigen is dependent on the disease to be treated.
  • the expansion antigen added to the first expansion step is a preferably TAA.
  • the disease to be treated is an infectious disease
  • the expansion antigen added in the first expansion step is a PAA.
  • the expansion antigen is preferably an autoantigen.
  • the expansion antigen in the first expansion step leads to a stimulation of clinically relevant lymphocytes within the cell mixture already at an early stage and thus together with the cytokine cocktail enhances the expansion of clinically relevant lymphocytes.
  • This stimulation is also referred to as antigen activation or antigen editing.
  • the first expansion step is followed by a second expansion step wherein the cells are incubated with feeder cells and/or an antibody against CD3 in addition to the cytokines IL-2, IL-15 and IL-21.
  • An expansion with feeder cells and the antibody against CD3 has been described in the state of the art. It is believed that feeder cells lead to an improvement of cell growth.
  • Feeder cells are irradiated cells that do not proliferate or proliferate only to a small extent.
  • the feeder cells increase the number of cell contacts in the culture and additionally feed the proliferating and expanding cell culture.
  • Feeder cells are preferably in irradiated PBMCs. Allogeneic feeder cells are from a different organism as the mammal to be treated with the expanded clinically relevant lymphocytes. Autologous feeder cells are from the mammal to be treated.
  • the antibody against CD3 is preferably the antibody defined as OKT3.
  • OKT3 is a murine monoclonal antibody of the immunoglobulin IgG2a isotype.
  • the target of OKT3, CD3, is a multi-molecular complex found only on mature T-cells. An interaction between T-cells, OKT3 and monocytes causes T-cell activation in vitro.
  • feeder cells are used in combination with CD3 and the cytokines IL-2, IL-15 and IL-21.
  • the ratio of feeder cells to lymphocytes is in the range of 1:1 to 1:100.
  • the ration of feeder cells to lymphocytes is in the range of 1:2 to 1:50.
  • very low ratios of feeder cells are sufficient for a strong expansion of clinically relevant lymphocytes, in particular clinically relevant T-cells.
  • a ratio of 1:10 is sufficient to support growth and expansion of lymphocytes. Accordingly it is believed that a value in the range of 1:5 to 1:20 would not lead to a different result.
  • the low number of feeder cells has at least two advantages. First, there are less distracting cellular signals allowing more homogeneous and reliable expansion results. Secondly, fewer feeder cells lead to less exogenous material in the immunotherapy product obtained by the method, i.e. the population of the clinically relevant lymphocytes.
  • the second expansion step optionally also includes an expansion antigen in the culture medium. Preferably no clinically relevant antigen or fragment is added to the medium of the second expansion step.
  • the method comprises a refocusing step.
  • the refocusing step comprises a culturing in culture medium comprising refocusing cells.
  • Refocusing cells are cells from the mammal, in particular a human, from which the body sample is obtained.
  • the refocusing cells are autologous cells that have been treated with at least one refocusing antigen.
  • Any expansion antigen as defined herein can also be used as refocusing antigen.
  • the one or more refocusing antigens are identical to the one or more expansion antigens.
  • Refocusing cells are cells that have been incubated with the refocusing antigen for at least 30 minutes, or more, for example at least one hour, at least two hours, at least five hours, or at least ten hours. After incubation with the refocusing the refocusing cells are irradiated with at least 40 Gy. Preferably the cells are irradiated with at least 45 Gy, or more, for example at least 50 Gy and particularly preferred with an intensity of 55 Gy. Due to this treatment, antigen-specific T-cells are more effectively expanded, recognize tumor, pathogens or auto-immune cells and may confer protection against cancer cells or pre-malignant lesions.
  • the time of the refocusing step is in the range of 1 to 6 days, preferably 1 to 3 days.
  • the refocusing step can be rather short, it leads to significant improvement in the yield of expanded clinically relevant lymphocytes, in particular for clinically relevant lymphocytes expanded from peripheral blood.
  • the number of refocusing cells in comparison to the number of lymphocytes is rather low.
  • the ratio of refocusing cells to lymphocytes is in the range of 1:1 to 1:100. It is found that the best results are achieved if the refocusing step is performed right after the first expansion step and followed by the second expansion step.
  • the sequence of culturing steps is in particular useful for generating a population of clinically relevant lymphocytes, in particular T-cells.
  • the first expansion step comprises adding IL-2, IL-15 and IL-21 simultaneously to the cell culture.
  • IL-2, IL-15 and IL-21 simultaneously to the cell culture.
  • the simultaneous application of IL-2, IL-15 and IL-21 leads to a preferred lymphocyte composition of the expanded lymphocyte population.
  • IL-15 and IL-2 may be added simultaneously or sequentially.
  • IL-15 is added secondly and IL-2 added last.
  • IL-15 is added as the first cytokine, followed by simultaneous addition of IL-2 and IL-21 or a sequential addition of IL-2 and IL-21.
  • IL-21 is added secondly and IL-2 is added last.
  • the culture medium of the first and/or second expansion step may comprise least one expansion antigen.
  • the expansion antigen may for example be fragment of a known TAA.
  • Possible TAAs for as expansion antigen are for example NY-ESO-1, tyrosinase tumor antigen, tyrosinase related protein (TRP)-1, TRP-2, VEGFR-2, and a member of the MAGE family of proteins, telomerase, p53, HER2/neu, mesothelin, carcinoembryonic, survivin, EGFRvIII, VEGF, CAMPATH 1-antigen, CD22, CA-125, Mucin-1, Alpha-1-getoprotein, PSMA.
  • TAAs for as expansion antigen are for example NY-ESO-1, tyrosinase tumor antigen, tyrosinase related protein (TRP)-1, TRP-2, VEGFR-2, and a member of the MAGE family of proteins, telomerase, p53
  • the fragment of the TAA is in particular a peptide.
  • Such peptide can be for example a peptide comprising at least eight continuous amino acids of an amino acid sequence that is at least 80 % identical to the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7.
  • SEQ ID NO: 4 is the amino acid sequence of the known tumor associated antigen NY-ESO-1.
  • SEQ ID NO: 5 is the amino acid sequence of the known tumor associated antigen survivin.
  • SEQ ID NO: 6 is the amino acid sequence of the known tumor associated antigen mesothelin.
  • SEQ ID NO: 7 is the amino acid sequence of the tumor associated antigen EGFRvIII.
  • the expansion antigen may for example be fragment of a known PAA.
  • Possible PAAs as expansion antigens are for example CMVpp65, or EBV (EBNA-3, EBNA-1), HPV-16/33 E6, E7 or L1.
  • the fragment of the PAA is in particular a peptide.
  • Such peptide can be for example a peptide comprising at least eight continuous amino acids of an amino acid sequence that is at least 80 % identical to the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, which are the amino acid sequence of the known pathogen associated antigens CMVpp65, EBNA-3, EBNA-1, and HPV-L1, respectively.
  • the expansion antigen may for example be fragment of a known PAA.
  • Possible autoantigens as expansion antigens are for example PRDM2, UCHL3, INO80E, SLC12A6, and Reelin.
  • the fragment of the PAA is in particular a peptide.
  • Such peptide can be for example a peptide comprising at least eight continuous amino acids of an amino acid sequence that is at least 80 % identical to the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, which are the amino acid sequences of the autoantigens PRDM2, INO80E, UCHL3, and DNaseB, respectively.
  • Additional components such as further cytokines may be added during the culturing step in particular during the first or second expansion step or the refocusing step.
  • a promoter compound may be added in the culturing step, in particular in the first or second expansion step or in the refocusing step.
  • the promoter compound is added in the first expansion step.
  • a promoter compound as used herein is a compound that in the expansion process causes an increase a specific subset of lymphocytes.
  • a preferred promoter compound is Zoledronic acid. Zoledronic acid promotes the expansion of gamma delta T-cells.
  • a further preferred promoter compound is promotes the expansion of B-cells
  • a co-stimulatory compound is added.
  • a co-stimulatory compound is for example a ligand for CD28 mediating T-cell signals.
  • ligands for CD28 mediating T-cell signals are members of the B7 superfamily,in particular B7-1 (CD80) and B7-2 (CD86).
  • testing for the presence of clinically relevant lymphocytes in the expanded lymphocyte sample comprises using evaluation antigens.
  • an evaluation antigen may be an antigen under the definition of the expansion antigen.
  • a known clinically relevant antigen or fragment thereof is added as evaluation antigen to the culture medium of the lymphocyte population to stimulate the lymphocytes. After an incubation time a parameter indicative for the activation of clinically relevant lymphocytes is measured.
  • the evaluation antigen is added to the lymphocytes in a form bound to a MHC I complex.
  • evaluation antigens may be presented to the lymphocytes bound to MHC dextramers.
  • MHC dextramers are fluorescently labeled MHC multimers bound to a dextrose backbone.
  • multimeric MHC structures has the advantage that multiple copies of the antigen can be presented to a single lymphocyte thereby increasing the simulation by the evaluation antigen.
  • known clinically relevant antigens may be presented to the cultured sample as evaluation antigens in the form of cells expressing the clinically relevant antigen as a transgene.
  • the parameter indicative for the presence of clinically relevant lymphocytes can be for example the production of one or more cytokines, in particular IFN- ⁇ or TNF ⁇ production.
  • Further parameters indicative of the presence of clinically relevant lymphocytes are an increased cell proliferation, an increased cytotoxicity, increased cell signaling and/or intracellular phosphorylation. The determination of these parameters is known in the art and exemplified in the examples.
  • evaluation antigens derived from known clinically relevant antigens it is also possible to test for clinically relevant lymphocytes specific for the mammal to be treated.
  • cells, in particular tumor cells, that are derived from the same mammal as the expanded lymphocytes are used for presenting the evaluation antigens.
  • evaluation presenting antigens the presence of clinically relevant lymphocytes specific for clinically relevant antigens that are not necessarily known clinically relevant antigens can be verified.
  • the evaluation antigens presented to the cultured sample are in a form selected from cells, in particular tumor cells, derived from the same mammal as the cultured sample (autologous cells) at least partially genetically matched allogeneic cells, in particular tumor cells or cell expressing the clinically relevant antigens as a transgene.
  • the method of the testing for the presence of clinically relevant lymphocytes comprises contacting the lymphocytes with at least one evaluation antigen and determining a change in either one of cytokine production, in particular IFN- ⁇ or TNF ⁇ production, cell proliferation, cytotoxicity, signaling and/or intracellular phosphorylation.
  • the testing of these parameters can be combined with flow cytometry and cell sorting. Accordingly, it is also possible to isolate the clinically relevant lymphocyte population, in particular the tumor reactive lymphocyte population from the expanded lymphocyte population.
  • the isolated population of clinically relevant lymphocytes may further be cultured or directly used for immunotherapy.
  • the method of the second aspect of the invention leads to the formation of population of lymphocytes, which includes a population of clinically relevant lymphocytes.
  • Clinically relevant lymphocytes included in the expanded lymphocyte population can be any type of lymphocyte.
  • Lymphocytes include B-cells, NK-cells and T-cells. According to one embodiment the clinically relevant lymphocyte is a B-cell. According to one embodiment the clinically relevant lymphocyte is an NK-cell.
  • the T-cell is preferably selected from a helper T-cell (T H -cell or CD4 + -T-cell), in particular a T H1 -cell, a cytotoxic T-cell (T C -cell or CD8 +- T-cell), in particular CD8 + CXCR3 + T-cell, a memory T-cell, in particular a central memory T-cell (T CM -cell), a stem cell memory T-cell (T SCM -Cell) or peripheral memory cell (T PM -cell), a gamma-delta T-cell ( ⁇ -T-cell), a NK-T-cell, a Mucosal-associated invariant T-cell (MAIT), a double-negative T-cell (CD3 + CD4 - CD8 - T-cell).
  • a helper T-cell T H1 -cell
  • T C -cell or CD8 +- T-cell cytotoxic T-cell
  • CD8 + CXCR3 + T-cell CD8 +
  • the clinically relevant lymphocyte may be a lymphocyte that expresses molecules that facilitate entry into tissues, in particular tumor or infected or inflamed tissue (e.g. CXCR3).
  • the clinically relevant lymphocyte is a lymphocyte that is enriched for markers of any long-term memory, in particular CD117 and c-kit and cytolytic immune cell responses, in particular CD107a.
  • the expanded lymphocyte population not only contains clinically relevant lymphocytes but also other lymphocytes that do not recognize clinically relevant antigens.
  • the invention relates to a lymphocyte obtained by a method according to the second aspect that expresses molecules and cytokines promoting the formation of combination of lymphocytes useful for medical application, including immunotherapy.
  • the combination of lymphocytes useful for medical application preferably comprises T-cell precursors, T CM , T SCM and/or T PM cells.
  • the population of lymphocytes comprises lymphocytes expresseing molecules and cytokines that promote the expansion of clinically relevant lymphocytes.
  • the lymphocyte obtained by the method according to the second aspect produces cytokines selected from IFN- ⁇ , TNF- ⁇ , IL-2, IL-17 and any combination thereof.
  • the lymphocyte is a CD3+CD4-CD8-T-cell.
  • the present invention provides a population of lymphocytes obtained by the second aspect of the invention comprising a population of clinically relevant lymphocytes,
  • the population of lymphocytes may consist of a population of clinically relevant lymphocytes.
  • the population of clinically relevant lymphocytes may be monoclonal, oligoclonal or polyclonal.
  • the population of clinically relevant lymphocytes is polyclonal and responds to multiple antigens or to different epitopes of the same antigen.
  • the population of clinically relevant lymphocytes responds to different antigens.
  • Lymphocytes in particular T-cells responding to multiple antigens may prevent the risk of recurrence of pre-malignant cells, tumor cells and pathogens, thereby decreasing the risk for immune escape.
  • the population of lymphocytes according to the third aspect has a composition of different lymphocyte phenotypes, in particular T-cell phenotypes that is beneficial for immunotherapy.
  • the population of lymphocytes has a low percentage of regulatory T-cells. Regulatory T-cells are known to suppress the therapeutic function of the population of lymphocytes. According to one embodiment of the third aspect in the population of lymphocytes, the percentage of T reg based on the total number of T-cells is below 5 %, preferably below 3 %.
  • the majority of T H cells in the population of lymphocytes comprise the T H1 phenotype.
  • the percentage of T H1 cells based on the total number of T H cells is at least 10 %, preferably at least 50 %, more preferably at least 70 %.
  • the percentage of the cytotoxic CD8+ T-cells is increased as could be shown that increased percentage of CXCR3+ cells.
  • the percentage of CXCR3+ cells based on the total number of CD8+ T-cells is at least 10 %, at least 50 %, more preferably at least 70 %.
  • the population of lymphocytes may comprise an increased number of T-cells that have recently been in contact with its antigen as for example identified by the 4-1BB+ phenotype.
  • the percentage of 4-1BB+ T-cells based on the total number of T-cells is at least 1 %, preferably at least 2 %, more preferably at least 2.5 %.
  • the percentage of CD107+ cells based on the total number of T-cells is at least 1 %, preferably at least 2 %, more preferably at least 2.5 %.
  • CD117+ T-cells have been associated with long-term memory population.
  • the CXCR3+ phenotype CD8+ T-cells is further associated with invasiveness into tissues and thus maybe in particular beneficial for immunotherapy against cancer. Further the population of lymphocytes may comprise sufficient percentage of CD3+CD4-CD8- cells. These double negative T-cells are highly specific for the antigen target as they are dependent of the co-receptor CD4+ or CD8+ and produce inflammatory cytokines. Thus, they are cytotoxic.
  • a population of lymphocytes comprises a percentage of CD3+CD4-CD8- cells based on the total number of T-cells of at least 1 %, preferably at least 3 %, more preferably at least 5 %.
  • the percentage of gamma delta T-cells based on the total number of T-cells in a population of lymphocytes is at least 1 %, preferably at least 3 %, more preferably at least 5 %.
  • Gamma delta T-cells have the effect that they recognize stressed cells, transformed cells, infected cells, e.g. CMV+ target cells.
  • Gamma delta T-cells cross - recognize as well virally and transformed cells.
  • the percentage of precursor T-cells (CD45RA+ CCR7+) based on the number of total T-cells is at least 1 %, preferably at least 2 %, more preferably at least 3 %.
  • Central memory T-cells were already shown to relate to success of a T-cell therapy.
  • the central memory cells produce several cytokines beneficial for therapy, provide a good memory response. However, the central memory cells do not infiltrate into tissue well.
  • the percentage of peripheral memory cells (CD45RA- CCR7-) based on the total number of T-cells at least 2 %, preferably at least 5 %, more preferably at least 10 %.
  • Peripheral memory cells show high cytokine production and infiltrate well into tissue. Thus, these cells are in particular useful for immunotherapy against cancer.
  • terminally differentiated T-cells are able to enter into a tissue. Moreover, these cells show a production of therapeutically useful cytokines.
  • the percentage of effector T-cells (CD45RA+ CCR7-) based on the total number of T-cells is at least 1 %, preferably at least 3 %, more preferably at least 5 %.
  • the percentage of precursor T-cells (CD45RA+ CCR7+) based on the total number of T-cells may be at least 1 %. Preferably, the percentage is at least 2 %, more preferably at least 3 %.
  • Precursor T-cells show only cytokine production and barely enter in tissue. However, these cells may be reverted T-cells with a lifelong memory effect.
  • the population of lymphocytes according to the third aspect may be further characterized by an intracellular cytokine production after stimulation with a evaluation antigen having a value that is at least twofold of the standard deviation without evaluation antigen stimulation. Moreover, the stimulation with the evaluation antigen leads to a value of CD107a induction that is at least twofold of the standard deviation without evaluation antigen stimulation.
  • lymphocytes and/or the population of clinically relevant lymphocytes represent useful immunotherapy products for the treatment of cancers, infectious diseases and autoimmune diseases.
  • the invention provides a composition comprising a population of clinically relevant lymphocytes according to the third aspect of the invention for use in medical treatment, in particular for treating and preventing an infectious disease, an autoimmune disease or a tumor disease.
  • the population of clinically relevant lymphocytes is preferably generated with the method according to the second aspect of the invention
  • composition for use according to the fourth aspect comprises an immunotherapy as defined below.
  • the immunotherapy comprised in the use of the composition may comprise the steps generating a clinically relevant lymphocyte population according to the second aspect of the invention wherein the body sample is obtained from said mammal and administering the clinically relevant lymphocyte population to said mammal.
  • the infectious disease may be any known infectious disease relating to any known pathogen.
  • the tumor disease according to the invention can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, cervical cancer, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, peritoneum, omentum, mesentery cancer, pancreatic cancer
  • the cancer patients may "conditioned" before administering the T-cells.
  • the conditioning has three purposes. First is to provide more space for the infused lymphocyte population, second to remove adverse effectors and third to increase production of growth factors that may favor the rapid expansion and survival of T-cells, i.e. autologous IL-7 and IL-15 production.
  • the clinically relevant lymphocytes can be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition, which is also within the scope of the present disclosure.
  • the clinically relevant lymphocytes may be autologous to the subject, i.e., the clinically relevant lymphocytes are obtained from the subject in need of the treatment, and then administered to the same subject.
  • the host cells are allogeneic cells, i.e., the cells are obtained from a first subject and administered to a second subject that is different from the first subject but of the same species.
  • allogeneic clinically relevant lymphocytes may be derived from a human donor and administered to a human recipient who is different from the donor.
  • an effective amount of the clinically relevant lymphocytes described herein, or compositions thereof can be administered to a subject (e.g., a human cancer patient, a patient with an infectious disease, a patient with an autoimmune disease) in need of the treatment via a suitable route, such as, for example, intravenous administration.
  • a suitable route such as, for example, intravenous administration.
  • the cells may be introduced by injection, catheter, or the like.
  • additional drugs e.g., cytokines
  • Any of the cells or compositions thereof may be administered to a subject in an effective amount.
  • an effective amount refers to the amount of the respective agent (e.g., the cells or compositions thereof) that upon administration confers a desirable therapeutic effect on the subject. Determination of whether an amount of the cells or compositions described herein achieved the desired therapeutic effect would be evident to one of skill in the art. For example, see references 2-5. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. In some embodiments, the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of the desired disease or disorder in the subject.
  • administering refers to the physical introduction of a composition comprising a cell or pharmaceutical composition described herein to a subject, using any of the various methods and delivery systems known to those skilled in the art.
  • the administering of the population of tumor reactive lymphocytes preferably is an introdution into the patient either locally in proximity of a tumor or into the tumor.
  • the population of tumor reactive lymphocytes is administered into the blood circuit.
  • Other clinically relevant lymphocytes are preferably added into the blood circuit.
  • the subject is administered a dose of clinically relevant lymphocytes comprising at least about, 4 x10 6 , 4.5 x10 6 , 5 x10 6 , 5.5 x10 6 , 6 x10 6 , 6.5 x10 6 , 7 x10 6 , 7.5 x10 6 , 8 x10 6 , 8.5 x10 6 , 9 x10 6 , 9.5 x10 6 , 10 x10 6 , 12.5 x10 6 , 15 x10 6 , 20 x10 6 , 25 x10 6 , 30 x10 6 , 35 x10 6 , 40 x10 6 , 45 x10 6 , 50 x10 6 , 60 x10 6 , 70 x10 6 , 80 x10 6 , 90 x10 6 of cells per kilogram body weight.
  • a dose of clinically relevant lymphocytes comprising at least about, 4 x10 6 , 4.5 x10 6 , 5 x10 6 ,
  • composition for use according to the fourth aspect administering the population of clinically relevant lymphocytes
  • the invention is further defined by the following examples.
  • Example 1 Lymphocytes expansion protocol using peripheral blood mononuclear cells
  • Aphaeresis was performed on a healthy donor primed with NY-ESO-1. After separation of the blood components the product containing the leucocytes is separated from the rest. Cells are suspended in Cellgro comprising 5 % pooled human AB serum, 1000 U/ml IL-2, 10 ng/ml IL-15 and 10 ng/ml IL-21. The medium in addition contained 10 ⁇ mol NY-ESO-1 peptide as expansion antigen. The concentration of cells from. The expansion of lymphocytes from peripheral blood contained the step of stimulation by irradiated autologous PBMCs pulsed with the antigen of interest.
  • autologous PBMCs were pulsed for two hours with 10 mM NY-ESO-1 peptide at RT, and then irradiated at 55 Gy.
  • the pulsed irradiated autologous PBMCs were added to the lymphocyte culture on day 7 and re-suspended in fresh culture medium supplemented with 5% human pooled AB serum with cytokines in concentrations as defined above.
  • On day 10 cells were expanded with Cellgro supplemented with 5 % pooled human AB serum and cytokines.
  • OKT3 antibody was added in a concentration of 30 ng/ml in the same culture medium described as above plus irradiated feeder cells (55Gy) at a 1:10 (feeder cell: T-cell ratio) the peptide and cytokines. On day 17 to 20 of culturing the cells are harvested for analysis or transferred to the patient.
  • Example 2 The procedure of Example 2 is identical to the procedure of Example 1 only that the donor was a patient who received vaccination with NY-ESO-1.
  • Example 3 is identical to Example 1 only that the donor is a patient whose tumor already expressed the TAA NY-ESO-1.
  • Example 4 - gamma delta T-cells can enriched in expanded lymphocytes with the cytokines IL-2, IL-15 and IL-21
  • the expansion is carried out according to the protocol described in Example 1 with the difference that zoledronic acid was added to the cell culture in a concentration of 5 ⁇ Mol.
  • Zoledronic acid is known to promote the expansion of TCR gamma delta T-cells.
  • this experimental setup leads to a strong increase of TCR gamma delta T-cells in both frequency and absolute numbers.
  • flow cytometry images of the expanded culture at different times are shown. Accordingly on the first day of expansion 3.68 % of he cells carry the TCR gamma delta. There is already a slight increase on the second day. Strikingly, after 7 days of expansion almost 50 % of T-cell are the ⁇ subset.
  • Example 5 Expansion of the lymphocytes with the cytokine cocktail induces double negative T-cells (CD3+CD4-CD8-).
  • Lymphocytes were obtained from a patient with narcolepsy. The expansion was carried out as described in Example 1 but with the PRDM2 peptide as expansion antigen and stimulating. The cultured cells were tested for the T-cell phenotype on day 1 and day 18.
  • Fig. 2 shows the flow cytometry results of these samples. In the results first the lymphocytes are filtered and then the CD3+ lymphocytes are filtered. These cells are then further analyzed for the presence of CD4 and CD8. From the panels it can be seen that after day 1 only 13 % of the cells are in the double negative state. The majority of the cells are CD4+. After 18 days of expansion with the cytokine cocktail 92 % of the cells are double negative cells (CD3+CD4-CD8-) T-cells.
  • the induction of IFN- ⁇ production of the expanded lymphocytes upon PRDM2 peptide stimulation is tested. As shown in Fig. 3 on day 1 only 0.89 % of the cells produce IFN- ⁇ upon PRDM2 stimulation. In contrast, the sample from day 18 shows a significantly increased population of cytokine producing PRDM2 specific T-cells. As most of the T-cells are in the CD3+CD4-CD8- state, this shows that the expansion protocol leads to the formation of clinically relevant double negative T-cells.
  • Example 6 Sorted Dextramer cells produce IFN- ⁇ in response to autologous EBV-LCLs loaded with peptide and protein antigens
  • the expansion protocol according to Example 1 was carried out with blood from two different patients with narcolepsy again with PRDM2 as expansion peptide.
  • DNAseB or PRDM2 peptides were added to the cytokine cocktail in the culture medium.
  • CD3+CD4-CD8- and conventional CD8+ T-cells that were MHC restricted and directed against PRDM2 or DNAseB were sorted by flow cytometry and tested whether they recognize naturally processed and presented epitopes.
  • T-cells from the same patients were pulsed with peptides or with recombinant proteins and IFN- ⁇ production was measured. The results of the experiment are summarized in Table 1:
  • the measured values are the IFN- ⁇ concentration in pg/ml.
  • This experiment shows that the cytokine cocktail of IL-2, IL-15 and IL-21 in combination with a specific antigen is able to expand clinically relevant T-cells, in particular T-cells specific for the antigen in peripheral blood.
  • the T-cells can preferentially reside in the so-called DN CD3+CD4-CD8- T-cell population and these T-cells are highly functionally active, they produce IFN- ⁇ and recognize biologically and medically relevant targets.
  • Example 7 Analysis of lymphocytes expanded with the cytokine cocktail and tumor associated antigens
  • Figs. 4 and 5 show the results of the flow cytometry.
  • Fig. 4A shows the signal of the cells separated by sideward and forward scattering. Lymphocytes are gated as shown by the black elliptic circle. The filtered lymphocytes are then again filtered for the presence of CD3. The CD3+ cells were further analyzed by a separation according to the presence or non-presence of CD8 and CD4.
  • Fig. 6A to 6C again shows the gating of CD8+ IFN- ⁇ producing T-cells.
  • the analysis of cells producing CD107a, CD127 (IL-7R) and CD117 are shown in Fig. 6D to 6F .
  • Grey signals stand for the entire CD3+CD8+ T-cell population (irrespective of the antigen-specificity), , black signals for CD3+CD8+ T-cells that are antigen-specific.
  • TAA reactive CD8+ T-cells express CD107a, a marker associated with ccytotoxicity and CD127 (the II-7R receptor, mediating survival signal signals), but not CD117, a marker for T-cells with stem-cell like properties.
  • PBMCs were obtained from a patient with glioblastoma. and were expanded with the cytokine cocktail of IL-2, IL-15 and IL-21 according to Example 1 but in combination with CMVpp65 peptide.
  • This experiment shows that the CMVpp65 peptide stimulation in combination with the cytokine cocktail of IL-2, IL-15 and IL-21 leads to a strong expansion of high affinity antigen specific T-cells (tetramer analysis). Results are shown in Fig. 7A to 7D .
  • APC-CMV indicating. PE-CMV und FITC-CMV stand for i) identical MHC class I-HLA-0201 molecules, ii) identical peptides. But the MHC molecule is mutated.
  • the CMV tetramers are differently mutated.
  • APC-CMV medium affinity, mutation in the position 245; between PE-CMV (high affinity) und FITC-CMV (wild type, comparably low affinity).
  • the data show that mutant tetramers that only allow high affinity T-cell receptors for binding detect high affinity T-cells (as well as T-cells with low and intermediate affinity) High affinity T-cells are believed to mediate stronger immune effector functions and to be superior in removing tumor cells and/or pathogens.
  • Example 9 Analysis of the frequency of tumor reactive T-cells after expansion of lymphocytes from peripheral blood with the cytokine cocktail and NY-ESO-1
  • PBMCs were obtained from a patient with pancreatic cancer. The experiment was carried out as described in Example 1.
  • Fig. 8 shows a flow cytometry analysis of samples of the expansion on day 0 and day 18. While on day 1 the IFN- ⁇ production upon NY-ESO-1 stimulation is only 1.85, this concentration increases drastically to 9.25 on day 18 (see Fig. 8B and 8D ). Thus the number of NY-ESO-1 specific T-cells is drastically increased during expansion.
  • Example 10 Expansion of survivin reactive T-cells from peripheral blood from patients with cancer
  • PBMCs were obtained from a patient with glioblastoma and were expanded with the cytokine cocktail of IL-2, IL-15 and IL-21 according to Example 1 but together with the known TAA survivin. Again cells were analyzed on day 1 and after 18 days of culture using flow cytometry. Before analysis the expanded cells were stimulated with survivin. Cells were grouped into CD4+, CD8+ and double negative T-cells. In these groups the concentration of IL-2, IFN- ⁇ and TNF- ⁇ was determined.
  • Fig. 9A shows the results for CD4+ cells
  • Fig. 9B the results for the double negative
  • Fig. 9C the results for the CD8+ cells.
  • T-cell responses there are no detectable T-cell responses, defined by cytokine production at time point 0 (T0).
  • T0 time point 0
  • cytokine production is found in response to stimulation by survivin proving the presence of survivin specific T-cells.
  • T-cell responses are generally very difficult to induce against survivin.
  • PBMCs were obtained from a patient with glioblastoma. The expansion was carried out as described in Example 1. Samples from day 0 and day 18 were analyzed according to the presence of CD45RA and CCR7 using flow cytometry. The results are shown in Fig. 10A and Fig. 10B .
  • cells were grouped into the following groups. Positive signals for CD45RA and CCR7 upper right section of the graph stands for precursor cells. CCR7+ CD45RA- lower right is central memory cells. CD45RA positive signal with CCR7- upper left area are the effector cells and the negative signal in both regions lower left area are the peripheral memory cells. As shown in comparison of Fig. 10A and Fig.
  • Example 12 Analysis of the differentiation of different T-cell subsets upon expansion with the cytokine cocktail of IL-2, IL-15 and IL-21 together with different TAA
  • PBMCs from patients with pancreas cancer were treated according to Example 1 with different antigens: GPI which is the cell surface bound part of mesothelin, survivin and NY-ESO-1.
  • GPI which is the cell surface bound part of mesothelin, survivin and NY-ESO-1.
  • Table 2 Central memory CD45RA-CCR7+ Precursor CD45RA+CCR7+ Effector T-cells CD45RA+CCR7- Peripheral memory CD45RA-CCR7- T-cell population Antigen for stimu lation Time-point 0 After stimulation Timepoint 0 After stimulation Time-point 0 After stimulation Time-point 0 After stimulation Time-point 0 After stimulation
  • the T-cell subset to be analyzed is shown: CD4+, CD8+, CD4- and CD8-.
  • the antigen stimulus is defined.
  • the percentage of central memory, precursor, differentiated effector cells and peripheral memory cells is shown at day 0 and day 18 of the different experiments. Accordingly, some antigens such as mesothelin particularly drive the expansion of precursor cells and defined by CD45RA+ CCR7+ and central memory cells defined by CD45RA- CCR7+.
  • PBMCs from patient with glioblastoma were expanded according to the protocol of Example 1 with peptides of the antigens (EGRvlll, NY-ESO-1 or survivin).
  • the cells were stimulated with the same peptides analyzed by flow cytometry and the results are summarized in Table 3: Table 3 Patient 1 Patient 2 Patient 3 EGFRV NY-ESO-1 Survivin TIMEPOINT 0 EGFRVIN NY-ESO-1 Survivin TIMEPOINT 0 EGFRV NY-ESO-1 Survivin TIMEPOINT 0 Lymphocytes 71,2 60,3 72,7 83,2 74 48,8 51,9 68,2 71,4 67,1 62,8 85,1 DD3+ 76 54,6 75,9 46,4 65,4 60,8 60,9 48,9 57,7 63,8 66,5 53,8 CD4+ 11 27,4 11,1 42,5 35 63,4 61,2 47,8
  • the numbers are percentages of the T-cell populations in the parental CD4+, CD8+, or double negative phenotype for each of the respective antigens at time point 18 days with comparison to time point 0 (start of the expansion).
  • An enrichment of precursor T-cell subsets, defined by CD45RA+ CCR7+ associated with antigen and strong expression of c-kit (CD117) is detected.
  • C-kit is a marker for T-cells with stem-cell like features.
  • CD117+ T-cells have been associated to provide long-term memory population. Strong induction of the cytotoxicity/degranulation marker CD107a in stimulated T-cells is also observed.
  • Example 14 The expansion protocol with the cytokine cocktail and TAA peptide leads to 4-1BB and TIM-3 positive T-cells
  • PBMCs were derived from patients with pancreatic cancer and expanded according to Example 1. Again TAA peptides were derived from GPI, NY-ESO-1 and survivin. Using flow cytometry the presence of different markers was tested. The markers are: 4-1BB, CD25, CD127, CTLA-4, LAG3, PD1 and TIM3.
  • Table 4 The results are summarized in Table 4: Table 4 4-1 BB CD 25+ CD 127+ CTL A-4+ LAG 33+ PD 1+ TIM 3+ T0 Ag T0 Ag T0 Ag T0 Ag T0 Ag T0 Ag T0 Ag T0 Ag T0 Ag T0 Ag T0 Ag Patient A CD4+ GP1 0,03 0,33 6,47 37,00 66,70 36,80 0,01 0,01 0,16 0,18 8,22 26,10 0,00 0,02 NY-ESO-1 5,96 25,80 45,30 0,00 0,28 25,70 0,17 Survivin 2,17 32,10 37,50 0,01 0,24 25,80 0,61 CD6+ GP1 0,04 0,03 1,46 23,90 23,40 1,77 0,10 0,06 0,85 0,29 7,43 39,20 0,00 0,01 NY-ESO-1 0,15 39,50 2,14 0,10 0,41 44,60 0,03 Survivin 0,11 37,10 2,47 0,15 0,56 39,70 0,03 CD4- CD6- GP1 0.04 0,14 0,89 28,10 12,90 1,05 0,07 0,02 0,
  • T0 stands for the concentration of the individual subsets defined in column 2, prior to expansion (T0).
  • Ag stands for 18 days of antigenic stimulation and cytokine-driven expansion.
  • the 4-1BB values and TIM3 values are indicative for antigen experienced T-cells. It is found that in particular 4-1BB+ T-cells are strongly increased in some of the experiments and cellular subsets. For example in the CD4+ subset stimulation with NY-ESO-1 leads to an 200-fold increase in 4-1BB+ T-cells (from 0.03 to 5,96).
  • lymphocytes are separated according to the presence of CD4 or CD8+.
  • the numbers in the table refer to percentages of cells positive for the individual markers listed in the second column in the table at T0 or after 18 days of expansion (Ag). Again for most patients a strong induction of 4-1BB+ T-cells is found.
  • Example 16 Analysis of lymphocytes expanded from a patient with pancreatic cancer and from a patient with glioblastoma
  • PBMCs were obtained from a patient with pancreatic cancer and from a patient with glioblastoma.
  • the lymphocytes were expanded with the protocol according to Example 1.
  • the lymphocyte culture is analyzed by flow cytometry against a variety of markers.
  • the results are represented in Table 6: Table 6 Pane GBM T0 TH T0 TH CD3 64 69,9 70 88,6 CD4 59,1 31,2 64 11 CCR6 Neg 88,9 97,4 95,9 86,6 TH1 9,69 97,1 4,82 38,8 TH2 0,23 0 0,64 0,2 CCR6 Pos 10,1 2,03 3,25 12 TH1* 22,2 92,1 9,64 68,1 TH17 1,05 0 1,02 0,52 CCR7-CD45RA+ 14,5 13,8 2,69 2,06 CCR7+ CD45RA+ 12,1 0,25 49 61,9 CCR7+ CD45RA- 10,9 0,2 21,2 23,3 CCR7-CD45RA- 62,5
  • the first column it is indicated what subgroup of lymphocytes was filtered.
  • the second column the respective tested markers are listed. If no marker is listed this line represents the percentage of total number of cells with the filter marker CD3+ based on the total number of cells and the CD4+, CD8+ or double negative.
  • T0 stands for the culture prior to expansion and TH for the time of harvest at day 18.
  • the numbers represent the percentage of cells carrying the selected markers in column 2 or column 1 respectively. It has to be noted that majority of CD4+ cells after expansion is TH1 positive. In pancreatic cancer the numbers increase from 22.2 to 92.1 % of the CD4 cells. In glioblastoma the results are only little less pronounced from 9.64 to 68.1 % of the CD4 cells.
  • CD8+ T-cells after expansion 96.8 % in pancreas and 85.5 % in glioblastoma. Accordingly almost all of the CD8+ cells are CXCR3+. Moreover it is noted a shift away from terminally differentiated T-cells into precursor or central memory T-cell subsets.
  • Fig. 11 shows flow cytometry graphs of the experiment for the determination of TH17, TH1, TH1* and TH2 in the sample from the pancreas cancer patient First the cells are gated on CCR6 and then on CCR3 and CCR4 respectively.
  • Example 17 Expansion of memory T-cells specific for certain antigens in the absence of stimulation with these antigens
  • PBMCs from patients with pancreas cancer were expanded according to Example 1 with the tumor associated antigen NY-ESO-1.
  • the resulting cells were stimulated for four hours with the following tumor associated antigens: CMV, EBNA-3a, INO80E, mesothelin, NY-ESO-1, survivin und UCHL3.
  • CMV tumor associated antigen
  • EBNA-3a tumor associated antigens
  • INO80E mesothelin
  • NY-ESO-1 fetalin
  • survivin und UCHL3 Using flow cytometry the percentage of cytokine producing T-cells was determined. The numbers are represented in Table 7 for different T-cell subsets: CD8+, CD4+ and double negative. The following cytokines were tested: IFN- ⁇ , IL-2 and TNF.
  • peripheral blood was obtained from a patient who suffered from severe HPV disease (HPV33 and HPV56).
  • the stimulation was performed with the HPV L1 peptide and the cytokine cocktail comprising IL-2, IL-15 and IL-21.
  • the cells were stimulated with L1 peptide and measured by flow cytometry.
  • the results of this are shown in Fig. 12.
  • Fig. 12D to 12F show the gating for lymphocytes CD3+ and CD8+ respectively.
  • the rectangle marks the area showing cells that express cytokine CD107a, a marker for degranulation/cytotoxicity that is antigen-specific.
  • Fig. 12A the results obtained with the L1 peptide
  • Fig. 12B a positive control
  • Fig. 12C just medium as negative control. Accordingly there is an increase from 0.77 to 2.23 in the percentage of cells producing CD107a.
  • Example 19 T-cells expanded with the cytokine cocktail and tumor antigens recognize autologous tumor cells
  • PBMCs from a patient with glioblastoma were stimulated with NY-ESO-1 peptides and CMVpp65 peptides.
  • Peripheral blood from a patient with glioblastoma was treated according to the protocol of Example 4 wherein in two setups the stimulating peptide was either NY-ESO-1 or CMVpp65.
  • a cytokine production assay was performed using different antigens for stimulation or the autologous tumor cells. It was found that antigen-specific expansion of T-cells using peptides and the cytokine cocktail leads to expansion of T-cells directed against the stimulating target, yet also against the patient's own autologous tumor cells.
  • Example 20 Comparison of lymphocyte expansion with two different cytokine combinations and TAA stimulation
  • Fig. 13 shows strongly increased IFN- ⁇ production and thus concentration of antigen-specific lymphocytes after expansion with IL-2, IL15 and IL-21 in comparison to the other cytokine composition or no cytokines.
  • Example 21 Comparison of lymphocyte expansion with two different cytokine combinations and TAA stimulation
  • Fig. 14 shows strongly increased IFN- ⁇ production and thus concentration of antigen-specific lymphocytes after expansion with IL-2, IL15 and IL-21 in comparison to the other cytokine composition or no cytokines.
  • T-cells derived from PBMCs were cultured in the presence of the cytokine cocktail IL-2, IL-15 and IL-21 and Treg (regulatory T-cells) were identified prior to and after expansion of T-cells using flow cytometry. The results are shown in Fig. 15 .
  • Fig. 15 from left to right: the gating of the T-cells on CD4+ T-cells and then on CD25high designating the high expression of the IL-2 receptor on activated T-cells is shown. Then the cells were gated on II-2R (high CD125) cells and tested for expression of the IL-7Receptor (CD127) and Foxp3 (intracellularly).
  • a Treg cell is defined as CD4+CD25high, Foxp3+ and CD127-negative.
  • the number of Treg was initially low (0.07% in CD4+ T-cells) and was even lower after T-cell expansion (0.01%).
  • the data imply that the cytokine cocktail is superior in T-cell expansion since IL-2 is known to drive strong Treg expansion, it also underlines the synergistic effects of the IL-2/IL-15/IL-21 combination that blocks Treg expansion, while allowing anti-tumor directed T-cells to expand.
  • T-cells were expanded using the cytokine mix IL-2/IL-15/IL-21 along with the NY-ESO-1 antigens for 21 days.
  • Table 9 Note the strong preferential expansion of the VB7.2 family in the CD8+ T-cells expanded in the GRex flask, as well as the VB13.2 family in the CD8+ T-cells (also expanded in the GRex flask).
  • the data show that the presence of the antigen (NY-ESO-1) drives preferential expansion of individual TCR VB families - and shows also the diversity of the response driven by the antigen (not only by the cytokines). Focused, yet diverse TCR VB families suggest a diverse anti-target directed T-cell response.
  • T-lymphocytes have been expanded using the IL-2/IL-15/IL-21 and the NY-ESO-1 peptide mix.
  • Top panel: prior, bottom panel: after cytokine/TAA expansion T-cells have been cultured in the presence of IL-2/IL-15/IL-21.
  • a flow cytometic analysis has been performed before and after T-cell stimulation. From Left to right: i) gating on lymphocytes, ii) gating on CD3+ T-cells. iii) gating on CD4/CD8. Top right: PD1 expression on CD8+ activated T-cells (37% T-cells).
  • Cytokine / TAA-expanded T-cells show decreased PD1 expression on the CD4CD8+ T-cells.
  • the data show that the cytokine/peptide expanded T-cells exhibit decreased frequency of PD1 + T-cells on CD8+ T-cells - that implies that these expanded T-cells may show longer life-time and therefore superior efficacy against tumor cells.
  • a peptide cocktail consisting of 12 individual peptides from the L1 protein from HPV 33, along with the cytokine cocktail, was used to stimulate T-cells from patients with HPV+ lesions. At day 21, the T-cells were tested for cytotoxicity directed against each individual peptide.
  • autologous EBV-transformed B-cells were taken and pulsed with peptides 1-12, followed by a standard Cr51 assay. This assays measure the capacity of T-cells to kill specific targets. The results are shown Fig 16 .
  • T-cells from donor A recognize strongly peptides 11 and 12.
  • T-cells from d B reccognized strongly peptides 7-12 with a strong reactivity against peptide 11.
  • Example 26 CD117 expression before and after cytokine- and NY-ESO-1 driven expansion of PBMCs.
  • PBMCs were expanded with IL-2/IL-15/IL-21-mix in the presence of the tumor - associated antigen NY-ESO-1.
  • the cells were analyzed by flow cytometry before ( Fig. 18 ) and after expansion ( Fig. 19 ).
  • CD3+ T-cells are gated, then the CD3+ T-cells are gated on CD4+ and CD8+ T-cells.
  • CD117 + cells were then detected on CD8+ T-cells (top) and on CD4+ T-cells (bottom panel, left).
  • Middle panel CD45RA/CCR7 expression in CD8+ and CD4+ T-cells with a high population on CD45RA+ CCR7+ T-cells, representing precursor Tcells (middle panel).
  • CD117+ T-cells (blue labeled) reside in effector T-cells in CD8+ T-cells; CD117+ T-cells can be found in different T-cell populations in the CD4+ T-cell subsets.
  • the data implies that CD117 expression, a marker of T-cell 'stem-ness' present is present on different T-cell subsets.
  • CD117+ T-cells are advantageous to provide a source for long-term T-cell memory.
  • CD3+ T-cells are gated, then the CD3+ T-cells are gated on CD4+ and CD8+ T-cells.
  • Middle panel CD45RA/CCR7 expression in CD8+ and CD4+ T-cells with a high population on CD45RA+ CCR7+ T-cells, representing precursor T cells (middle panel).
  • CD84 T-cells CD117+ T-cells can be found in the T-cell precursor CD45RA+CCR7+ population.
  • the data implies that CD117 expression, a marker of T-cell 'stem-ness' present is present on different T-cell subsets.
  • CD117+ T-cells are advantageous to provide a source for long-term T-cell memory. It also shows that the cytokine cocktail strongly expands CD117+ T-cells that bring about long-term immune cell memory, also that CD117+ T-cells reside in precursor T-cells - to ensure long-term tumor immune responses.
  • This example describes the procedure for culturing TILs from pancreatic cancer for a function test and immunotherapy.
  • Example 1 For material, equipment and supplies see Example 1.
  • Tumor tissue obtained from a patient was put into a sterile container.
  • the tissue was cut into pieces of 1 to 2 mm 3 with a sterile scalpel.
  • the tissue pieces were put into the wells of 24 well plate with 1 piece per well.
  • Cell culture medium is prepared using Cellgro with 10 % human AB serum.
  • IL-2, IL-15 and IL-21 are added to a final concentration of 1000 u/ml for IL-2, 10 ng/ml for each of IL-15 and IL-21.
  • PEST and amphotericin were added to the medium. 1 ml medium was added into each well and the cell culture was incubated for 7 days at 37° Celsius.
  • PBMCs from a healthy donor were cultured in Cellgro containing 10 % human AB serum. The concentration of the PBMCs was determined and adjusted to a concentration of 10 6 /ml. The PBMCs were then radiated for 18 minutes at 55 Gy. After Irradiation OKT3 was added to a final concentration of 10 ng/ml. This culture is referred to as feeder cells with OKT3. On day 10 of the TIL culture, 100 ⁇ l of the feeder cell culture with OKT3 is added to each of the wells of the 24 well plate. Accordingly, the culture in each of the well to the final concentration of 10 ng/ml OKT3 and a total amount of 10 6 feeder cells. The ratio of TILs and feeder cells is about 1:10.
  • Example 28 Expansion of TIL culture from Pancreatic Cancer.
  • Tumor tissue obtained from a patient was put into a sterile container.
  • the tissue was cut into pieces of 1 to 2 mm 3 with a sterile scalpel.
  • the tissue pieces were put into the wells of 24 well plate with 1 piece per well.
  • Cell culture medium is prepared using Cellgro with 10 % human AB serum.
  • IL-2, IL-15 and IL-21 are added to a final concentration of 1000 u/ml for IL-2, 10 ng/ml for each of IL-15 and IL-21.
  • PEST and Amphotericin were added to the medium. 1 ml medium was added into each well and the cell culture was incubated for 4 days at 37° Celsius.
  • PBMCs from a healthy donor were cultured in Cellgro containing 10 % human AB serum. The concentration of the PBMCs was determined and adjusted to a concentration of 10 6 /ml. The PBMCs were then radiated for 18 minutes at 55 Gy. After Irradiation OKT3 was added to a final concentration of 10 ng/ml. This culture is referred to as feeder cells with OKT3. On day 10 of the TIL culture 100 ⁇ l of the feeder cell culture with OKT3 is added to each of the wells of the 24 well plate. Accordingly, the culture in each of the well contains the final concentration of 10 ng/ml OKT3 and a total amount of 10 6 feeder cells. The ratio of TILs and feeder cells is about 1:10.
  • Pancreas tumor tissue is obtained directly after operation and put into a sterile container.
  • the tumor tissue is cut into pieces of 1 to 2 mm 3 with a sterile scalpel. Each of the tissue pieces are to be transferred into a well of a 24 well plate.
  • 1 ml of the culture medium RPMI 1640 with 10 % fetal bovine serum was added into each of the wells.
  • the medium was changed on day 7 and day 14 which means that the culture medium was removed and replaced by fresh culture medium of the same kind.
  • the tumor cells reached a very high density the culture was transferred to a 6 well plate.
  • Fig. 20 A shows the lymphocyte culture of the one week of incubation. Some lymphocytes are detectable, however the concentration of lymphocytes is below the basic line of expansion.
  • Fig. 20 B and Fig. 20 C show the same lymphocyte culture after two weeks of incubation. Now the number of lymphocytes that can be detected has increased in this above the basic line of expansion. Feeder cells may be added at this stage.
  • Figs. 1 D, E and E show the culture four days, one week and two weeks subsequently after adding the feeder cells. In the figures can be seen that the number of lymphocytes drastically increases from the image in Fig. D to the image in Fig. F. In Fig. F it can be seen that the lymphocytes are gathering and attacking tumor cells in culture.
  • Example 31 Analysis of phenotype and activation/exhaustion marker expression of lymphocytes expanded from TIL obtained from Glioblastoma.
  • Tumor tissue was obtained from 16 Glioblastoma patients. TILs were expanded from the tissue with the cytokine cocktail of IL-2, IL-15 and IL-21 according to the protocol of Example 1.
  • the expanded cells were analyzed by flow cytometric analysis with regard to their CD4/CD8 phenotype using antibodies directed against CD3, CD4 and CD8.
  • TIL could be expanded from each individual tumor mainly contain CD3+ T-cells.
  • CD3+ T-cells Depending on the patient either one of CD4+, CD8+ and DN T-cell are in the majority. However, CD4+ was found most frequently in majority.
  • the table also shows that the cytokine cocktail is able to expand T-cells from different CNS tumor histologies. (see column 4 "Diagnosis")
  • the expanded cells were analyzed by flow cytometric analysis with regard to their specific phenotype - precursor (CD45RA+CCR7+), central memory (CD45RA-CCR7+), peripheral memory (CD45RA-CCR7-), or differentiated effector (CD45RA+CCR7-) T-cells and the expression of activation/exhaustion markers in the base phenotypes CD8+, CD4+ and double negative T-cells.
  • Fig. 22 A and B The results are summarized in Fig. 22 A and B .
  • the majority of CD8+, CD4+ or DN T-cells are in the central memory T-cell subset that has been shown to be crucial for effective anti-cancer responses and long-term immune memory.
  • the results shows in average a strong expansion of DN-T-Cells. This subset is highly activated and bears affinity T-cell receptors. Also a strong expansion of CD45RA+CCR7+ precursor T-cell subsets is observed that provide long-term memory T-cell responses advantageous for long-term immune surveillance.
  • the reported expression of 4-1BB, LAG-3 or TIM-3 in the T-cells are indicative for antigen/tumor specific T-cells.
  • the example shows that the cytokine cocktail expands TIL that reside primarily in the memory (the central memory) T-cell subset which has been associated with beneficial clinical responses, it also shows that TIL are expanded that bear markers associated with antigen-specificity.
  • Example 32 Analysis of phenotype and activation/exhaustion marker expression of lymphocytes expanded from TIL obtained from pancreas cancer
  • Tumor tissue was obtained from 17 pancreas cancer patients.
  • Table 12 summarizes the patient's age, sex, type of sample and histology.
  • Table 12 - The cytokine cocktail expands TIL from different pancreas cancer histologies.
  • TILs were expanded from the tissue with the cytokine cocktail of IL-2, IL-15 and IL-21 according to the protocol of Example 28.
  • the expanded cells were analyzed by flow cytometric analysis with regard to their CD4/CD8 phenotype using antibodies directed against CD3, CD4 and CD8. The results are summarized in Table 13. Table 13 T-cell phenotype analysis in pancreas cancer TIL. Dominant CD8+ TIL populations.
  • the expanded cells were analyzed by flow cytometric analysis with regard to their specific phenotype - precursor (CD45RA+CCR7+), central memory (CD45RA-CCR7+), peripheral memory (CD45RA-CCR7-), or differentiated effector (CD45RA+CCR7-) T-cells in the base phenotypes CD8+ and CD4+.
  • the results are summarized in Fig. 23 and 24 .
  • CD8+, CD4+ or DN T-cells are in the central memory T-cell subset that has been shown to be crucial for effective anti-cancer responses and long-term immune memory.
  • the results shows in average a strong expansion of DN-T-Cells (data not shown). This subset is highly activated and bears affinity T-cell receptors. Also a strong expansion of CD45RA+CCR7+ precursor T-cell subsets is observed that provide long-term memory T-cell responses advantageous for long-term immune surveillance.
  • the reported expression of 4-1BB, LAG-3 or TIM-3 in the T-cells are indicative for antigen/tumor specific T-cells.
  • the example shows that the cytokine cocktail expands TIL that reside primarily in the memory (the central memory) T-cell subset which has been associated with beneficial clinical responses, it also shows that TIL are expanded that bear markers associated with antigen-specificity.
  • Example 33 Analysis of the TCR length of T-cells expanded from tumor tissue of patients with pancreatic cancer
  • TILs were expanded from the tissue with the cytokine cocktail of IL-2, IL-15 and IL-21 according to the protocol of Example 28. Using a PCR-based approach the TCR length was measured. The 'normal' image of a TCR family is a Gauss-distribution of the length of the T-cell receptor. Single peaks suggest monoclonality in individual TCR families.
  • the data presented in Fig. 25 shows that the TIL composition is monoclonal or polyclonal, suggestive to be focused against autologous tumor targets, the cytokine cocktail aids to expand a focused TCR repertoire.
  • Example 34 Analysis of cytokine production in lymphocytes expanded from TIL obtained from patients with glioblastoma
  • Tumor tissue was obtained from glioblastoma patients.
  • TILs were expanded from the tumor tissue with the cytokine cocktail of IL-2, IL-15 and IL-21 according to the protocol of Example 1.
  • the expanded lymphocytes were stimulated with peptides derived from tumor - associated antigens, i.e. the EGRvrlll, NY-ESO-1 or survivin and the percentage of cells producing either one of the cytokines IFN ⁇ and TNF ⁇ was measured.
  • Fig 26 B The results are shown in Fig 26 B .
  • maximal stimulation is tested with PMA/inonomycin as a positive control and the background signal is determined with media only (negative control).
  • the results demonstrate that the expanded T-cells recognize these commonly shared tumor antigens.
  • the cytokine cocktail expands T-cells from glioblastoma that are reactive to tumor-antigens that have been described to be clinically relevant and associated with clinical responses.
  • Example 35 Analysis of cytokine production in lymphocytes expanded from TIL obtained from patients with pancreas cancer
  • Tumor tissue was obtained from pancreas cancer patients.
  • TILs were expanded from the tumor tissue with the cytokine cocktail of IL-2, IL-15 and IL-21 according to the protocol of Example 27.
  • the expanded lymphocytes were stimulated with peptides derived from tumor-associated antigens, i.e. the EGRvrlll, NY-ESO-1 or survivin and the percentage of cells producing either one of the cytokines IFN ⁇ and TNF ⁇ was measured.
  • the results are shown in Fig 27 A.
  • Fig. 27 B shows images of the flow cytometry analysis for NY-ESO-1. The results confirm that the expanded T-cells recognize these commonly shared tumor antigens.
  • the cytokine cocktail expands T-cells from pancreas tumor that are reactive to tumor-antigens that have been described to be clinically relevant and associated with clinical responses.
  • Example 36 Analysis of cytokine production in lymphocytes expanded from TIL obtained from patients with glioblastoma and autologous stimulation
  • Tumor tissue was obtained from glioblastoma patients.
  • TILs were expanded from the tumor tissue with the cytokine cocktail of IL-2, IL-15 and IL-21 according to the protocol of Example 27.
  • the expanded lymphocytes were stimulated with autologous tumor cells.
  • the results are shown in Fig 28 A.
  • Fig. 28 B shows images of the flow cytometry analysis. The results confirm that the expanded T-cells recognize autologous tumor cells.
  • the cytokine cocktail expands T-cells from glioblastoma that are reactive to autologous tumor cells, accordingly the patient's own mutations.
  • Example 37 Link of TCR usage in pancreatic cancer with recognition of autologous tumor cells
  • TILs were expanded from the tumor tissue with the cytokine cocktail of IL-2, IL-15 and IL-21 according to the protocol of Example 27.
  • the expanded lymphocytes were stimulated with autologous tumor cells.
  • TIL were first gated on CD3+ T-cells, then on CD4+ and CD8+ T-cells.
  • the frequency of individual V ⁇ families were tested using a panel of TCR VB antibodies. This TCR panel covers about 75% of the human TCR repertoire, there is therefore a possibility that not all TCR V ⁇ families are captured sufficiently.
  • the TCR V ⁇ family distribution is about 2 to 6 % in each family, except for TCR VB 2 that can reach over 10%.
  • Table 14 shows the preferential expansion of the V ⁇ -2 family in TIL from patients with glioblastoma.
  • TCR VB are preferentially expanded in TIL. It is noted that clonality can only be addressed by sequencing. It is noted that the cytokine cocktail expands different TCR V ⁇ families in individual patients. This is true for patients with glioblastoma, as well as for patients with patients with pancreatic cancer. Note also that some TIL are composed for a single or two VB families showing a highly focused TCR VB expansion. suggestive of an antigen-driven T-cell expansion process. Some TIL that we have shown to be preferentially expanded were shown to be monoclonal. Thus, the cytokine cocktail of IL-2, IL-15 and IL-21 expands a focused T-cell response, that is directed against the patient's own tumor cells.
  • Example 38 Link of TCR usage in pancreatic cancer with recognition of autologous tumor cells
  • Tumor tissue was obtained from pancreatic cancer patients. TILs were expanded from the tumor tissue with the cytokine cocktail of IL-2, IL-15 and IL-21 according to the protocol of Example 28.
  • IFN ⁇ production is measured after 3 days after exposure of TIL to autologous tumor cells (single cell suspension). High level of IFN ⁇ production is seen after with autologous tumor cells only (see Fig. 35) IFN ⁇ production is complete blocked with the antibody against W6/32 (blocking CD8+ TIL). As a control, the antibody L243 blocking CD4+ TIL is not able to block reactivity, the reactive T-cells in expanded cell lymphocytes are all CD8+. The data show that the cytokine cocktail expands TIL that are very focused and specifically recognized autologous tumor cells from patients with pancreatic cancer.
  • Example 39 Analysis of the cytolytic response of expanded TIL from patients with glioblastoma against autologous tumor cells
  • Tumor tissue was obtained from glioblastoma patients.
  • TILs were expanded from the tumor tissue with the cytokine cocktail of IL-2, IL-15 and IL-21 according to the protocol of Example 28.
  • Autologous tumor cells were generated according to the protocol in Example 29.
  • Autologous tumor cells are labeled with radioactivity and cultured with expanded TIL for 4 h. Killing of tumor cells leads to release of radioactivity which is then measured.
  • the principle of the method is shown in Fig. 21 .
  • the results are shown in Fig. 31 confirming a cytotoxic effect dependent on the ratio of TILs to tumor cells.
  • the data show that the cytokine cocktail expands TIL that are strongly cytoxic against autologous tumor cells.
  • expanded monoclonal T-cells and/or preferentially expanded TIL were tested for their cytotoxicity with the same test. The results are shown in Fig. 32 .
  • Table 16 shows that these immune responses are specifically directed against the autologous tumor cells.
  • the data shows that the cytokine cocktail expands TIL with a specific reactivity, including cytotoxic responses, against autologous tumor.
  • TIL that showed absent IFN ⁇ production were strongly cytotoxic, as measured in a standard CR51 release assay.
  • the data show that TIL produce IFNgamma against autologous tumor cells in a specific fashion.
  • Example 40 Analysis of the cytolytic response of expanded TIL from patients with pancreas cancer against autologous tumor cells
  • Tumor tissue was obtained from pancreatic cancer patients.
  • TILs were expanded from the tumor tissue with the cytokine cocktail of IL-2, IL-15 and IL-21 according to the protocol of Example 28.
  • Autologous tumor cells were generated according to the protocol in Example 29.
  • Autologous tumor cells are labeled with radioactivity and cultured with expanded TIL for 4 h. Killing of tumor cells leads to release of radioactivity which is then measured.
  • the principle of the method is shown in Fig. 7 .
  • an autologous (melanoma) - TIL system was used.
  • the results are shown in Fig. 33 confirming a cytotoxic effect.
  • the data show that the cytokine cocktail expands TIL also from patients with pancreatic cancer.
  • These TIL are highly focused - based on the TCR usage - and show a specific reactivity, including cytotoxic responses, against autologous tumor.
  • Tumor tissue was obtained from glioblastoma patients.
  • TILs were expanded from the tumor tissue with the cytokine cocktail of IL-2, IL-15 and IL-21 according to the protocol of Example 27. Cells were analyzed by flow cytometry for marker expression that defined T-cell function and homing.
  • the results of the analysis are summarized in Table 17.
  • the Th1 cells as well as the CXCR3 were less than 10% prior to expansion.
  • the data show that the cytokine cocktail leads to a T-cell product in which the CD8+ cells consists essentially of CXCR3+ CD8+ T-cells. This phenotype is enabled to enter into the tumor tissue.
  • the CD4+ T-cells almost all have a T H1 profile.
  • the T H1 profile IFNgamma and TNFalpha production leads to improved anti-tumor responses.
  • CD3+CD4-CD8- (DN) T-cells are present, a T-cell subset, associated with strong-autoimmune and tumor responses, which also expresses the marker CXCR3 that enables better penetration into tumor tissue.
  • Example 42 Recognition of commonly shared Tumor antigens by TIL from patients with pancreatic cancer
  • TAAs as 15 overlapping peptides were incubated for three days with TIL and IFN ⁇ production (pg/mL) was measured by ELISA.
  • antigen responses were either blocked by an antibody blocking MHC class I antigen responses (W6/32) affecting CD8+ T-cells, or blocking HLA-DR (MHC class II responses, L243) affecting CD4+ T-cells.
  • the IFN ⁇ production (pg/mL) in each of the samples is shown in Table 18.
  • IFN ⁇ production in TIL that is antigen-specific as shown by blocking with anti-MHC class I (blocking CD8+ T-cells) or with anti-MHC class II (blocking CD4+) T-cells.

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Claims (15)

  1. Composition pour l'expansion in vitro de lymphocytes comprenant l'interleukine 2 (IL-2), l'interleukine 15 (IL-15) et l'interleukine 21 (IL-21), dans laquelle la composition est sous forme liquide, la concentration en IL-2 dans la composition liquide est comprise dans la plage allant de 500 à 2 000 U/ml, la concentration en IL-15 est comprise dans la plage allant de 0,1 à 100 ng/ml et dans laquelle la concentration en IL-21 est comprise dans la plage allant de 0,1 à 100 ng/ml.
  2. Composition selon la revendication 1, dans laquelle la concentration en IL-2 dans la composition liquide est comprise dans la plage allant de 800 à 1 200 U/ml.
  3. Composition selon la revendication 2, dans laquelle la concentration en IL-15 est comprise dans la plage allant de 2 à 50 ng/ml, de préférence dans la plage allant de 5 à 20 ng/ml et dans laquelle la concentration en IL-21 est comprise dans la plage allant de 2 à 50 ng/ml, plus préférablement dans la plage allant de 5 à 20 ng/ml.
  4. Procédé de préparation d'une population de lymphocytes cliniquement pertinents, comprenant les étapes de :
    - fourniture d'un échantillon corporel obtenu auprès d'un mammifère, en particulier un échantillon de tissu ou un échantillon de liquide corporel, comprenant au moins un lymphocyte et éventuellement séparation des cellules dans l'échantillon corporel ;
    - culture in vitro de l'échantillon corporel pour l'expansion et/ou la stimulation des lymphocytes dans l'échantillon, la culture comprenant l'utilisation d'IL-2, d'IL-5 et d'IL-21 ; la concentration en IL-2 dans la culture étant comprise dans la plage allant de 500 à 2 000 U/ml ; la concentration en IL-15 étant comprise dans la plage allant de 0,1 à 100 ng/ml et la concentration en IL-21 étant comprise dans la plage allant de 0,1 à 100 ng/ml ;
    - et éventuellement détermination de la présence de lymphocytes cliniquement pertinents dans l'échantillon cultivé ;
    dans lequel la culture in vitro comprend une première étape d'expansion comprenant une incubation dans un milieu de culture comprenant de l'IL-2, de l'IL-5 et de l'IL-21 jusqu'à ce que les lymphocytes deviennent détectables, et dans lequel la culture in vitro comprend une seconde étape d'expansion comprenant une incubation dans un milieu de culture comprenant des cellules nourricières et/ou un anticorps anti-CD3 en plus de l'IL-2, de l'IL-5 et de l'IL-21.
  5. Procédé selon la revendication 4, dans lequel les lymphocytes cliniquement pertinents sont choisis parmi les lymphocytes réactifs aux tumeurs, les lymphocytes réactifs aux pathogènes et les lymphocytes réactifs aux maladies auto-immunes, de préférence les lymphocytes réactifs aux tumeurs.
  6. Procédé selon la revendication 4 ou 5, dans lequel l'échantillon corporel est choisi dans le groupe constitué par le sang périphérique, le sang de cordon ombilical, la moelle osseuse, les ganglions lymphatiques, le foie, l'épanchement pleural, le thorax, la cavité abdominale, le liquide synovial, le péritoine, l'espace rétropéritonéal, le thymus et la tumeur, de préférence le sang périphérique ou la tumeur.
  7. Procédé selon l'une quelconque des revendications 4 à 6, dans lequel le temps d'incubation de la première étape d'expansion est compris dans la plage allant de 6 heures à 180 jours, de préférence dans la plage allant de 4 à 10, plus préférablement dans la plage allant de 6 à 8 jours, de manière préférée entre toutes d'environ 7 jours.
  8. Procédé selon l'une quelconque des revendications 4 à 7, dans lequel le rapport des cellules nourricières aux lymphocytes est compris dans la plage allant de 1:1 à 1:100, de préférence dans la plage allant de 1:2 à 1:50, plus préférablement dans la plage allant de 1:5 à 1:20, de manière préférée entre toutes d'environ 1:10.
  9. Procédé selon l'une quelconque des revendications 4 à 8, dans lequel le milieu de culture de la première et/ou seconde étape d'expansion comprend au moins un antigène d'expansion.
  10. Procédé selon la revendication 9, dans lequel l'antigène d'expansion est un fragment de TAA, en particulier un peptide comprenant au moins huit acides aminés continus d'une séquence d'acides aminés qui est au moins 80 % identique aux séquences d'acides aminés SEQ ID NO : 4, SEQ ID NO : 5, SEQ ID NO : 6, SEQ ID NO : 7, SEQ ID NO : 8, SEQ ID NO : 9, SEQ ID NO : 10, SEQ ID NO : 11, SEQ ID NO : 12, SEQ ID NO : 13 ou SEQ ID NO : 14.
  11. Procédé selon l'une quelconque des revendications 4 à 10, dans lequel la culture in vitro comprend en outre une étape de recentrage, comprenant une incubation dans un milieu de culture comprenant des cellules de recentrage, dans lequel de préférence la durée de l'étape de recentrage est comprise dans la plage allant de 1 à 6 jours, de préférence de 1 à 3 jours.
  12. Procédé selon l'une quelconque des revendications 4 à 11, dans lequel l'analyse pour la présence de lymphocytes cliniquement pertinents comprend l'utilisation d'antigènes d'évaluation et les antigènes d'évaluation sont présentée à l'échantillon cultivé sous une forme choisie parmi les cellules, en particulier les cellules tumorales, dérivées du même mammifère que l'échantillon cultivé (cellules autologues), les cellules allogéniques au moins génétiquement partiellement appariées, en particulier les cellules tumorales ou les cellules exprimant les antigènes cliniquement pertinents en tant que transgène.
  13. Procédé selon la revendication 12, dans lequel l'analyse pour la présence de lymphocytes cliniquement pertinents comprend la mise en contact des lymphocytes avec au moins un antigène cliniquement pertinent et la détermination d'un changement de l'une quelconque parmi la production de cytokines, en particulier la production d'INFγ, la prolifération de cellules, la cytotoxicité, la signalisation et/ou la phosphorylation intracellulaire.
  14. Population de lymphocytes obtenue par un procédé selon l'une quelconque des revendications 4 à 13 comprenant une population de lymphocytes cliniquement pertinents, caractérisée par une ou plusieurs des caractéristiques suivantes :
    - le pourcentage de Treg sur la base du nombre total de lymphocytes T est inférieur à 5 %, de préférence inférieur à 3 % ;
    - le pourcentage de lymphocytes TH1 sur la base du nombre total de lymphocytes TH est d'au moins 50 %, de préférence d'au moins 70 %, plus préférablement d'au moins 80 %,
    - le pourcentage de lymphocytes T CXCR3+ sur la base du nombre total de lymphocytes T CD8+ est d'au moins 50 %, de préférence d'au moins 70 %, plus préférablement d'au moins 80 %,
    - le pourcentage de lymphocytes T 4-1BB+ sur la base du nombre total de lymphocytes T est d'au moins 1 %, de préférence d'au moins 2 %, plus préférablement d'au moins 2,5 %,
    - le pourcentage de lymphocytes T CD117+ sur la base du nombre total de lymphocytes T est d'au moins 1 %, de préférence d'au moins 2 %, plus préférablement d'au moins 2,5 %,
    - le pourcentage de lymphocytes CD3+CD4-CD8- sur la base du nombre total de lymphocytes T est d'au moins 1 %, de préférence d'au moins 3 %, plus préférablement d'au moins 5 % ; et
    - le pourcentage de lymphocytes T γδ sur la base du nombre total de lymphocytes T est d'au moins 1 %, de préférence d'au moins 3 %, plus préférablement d'au moins 5 %.
  15. Composition comprenant une population de lymphocytes selon la revendication 14, pour une utilisation dans un traitement médical, en particulier pour le traitement ou la prévention d'une maladie infectieuse, d'une maladie auto-immune ou d'une maladie tumorale.
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IL249409B (en) 2020-06-30
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CN106574244B (zh) 2022-01-07
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JP7413639B2 (ja) 2024-01-16
CA2951749A1 (fr) 2015-12-17
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US20170107490A1 (en) 2017-04-20
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